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Microscopes and Microscopy
MCB 380
Good information sources:
Alberts-Molecular Biology of the Cell
http://micro.magnet.fsu.edu/primer/
http://www.microscopyu.com/
Approaches to Problems in
Cell Biology
 Biochemistry-You can define a enzyme reaction and
then try to figure what does it, when, where and
under what control
 Genetics- You can make a mutation and then try to
figure out what you mutated
 Cell Biology- You can visualize a process and try to
understand it- for instance cell division was one of the
earliest
 Today- there are no distinctions. You cannot be just
one thing, or be knowledgable about one thing. You
need to take integrated appoaches to problems using
the appropriate tools when needed. If you limit your
approach, you limit your science
Properties of Light
 Reflection
 Diffraction-scattering of light around edges of objects
 Limits the resolution
 Refraction- bending of light when changing medium
(index of refraction)
 principle that lenses use to focus light
 Used in contrasting techniques
 Interference
 light waves can subtract and add
 Polarization- allowing only light of a particular
vibrational plane
Refraction
Diffraction
Constructive
Destructive
Interference
Limitations
 light waves diffract at edges-smearing
causes limits
 resolution = minimum separation of two
objects so that they can both be seen
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Limit of Resolution
 The cone of light collected by the lens
determines the resolution (nsinq)
n=refractive index
 Max NA is 1.4 (refractive index of oil)
 Lenses range from 0.4-1.4 NA
 Maximum magnification is about 1000x
Resolution = 0.61l/nsinq = 0.61l/NA
microscopy.ppt
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Resolution of Microscopes
 Visible light is 400-700nm
 Dry lens(0.5NA), green(530nm
light)=0.65µm=650nm
 for oil lens (1.4NA) UV light (300nm) =
0.13µm
 for electron microscope
 l=0.005nm but NA 0.01 so =30-50nm
Sizes of Objects
 Eukaryotic cell- 20µm
 Procaryotic cell-1-2µm
 nucleus of cell-3-5µm
 mitochondria/chloroplast- 1-2µm
 ribosome- 20-30nm
 protein- 2-100nm
Microscope Objectives
 complex combinations of lenses to
achieve
 high magnification
 low optical distortion
 Low chromatic distortion
 flat field
Contrast
 Cells are essentially water and so are
transparent
 In addition to resolution and brightness, you
need to generate contrast to see things
 Two objects may be resolvable by the
microscope, but if they don’t differ from the
background, you cannot see them
 Contrast can be accomplished with staining
or optical techniques
Microscope types
 Brightfield
 Stereo
 Phase contrast
 Differential Interference Contrast
 Fluorescence
 Confocal
 Electron
 Transmission
 Scanning
 Atomic Force
Microscopes
 Stereo
 Different images are sent to the two eyes from
different angles so that a stereo effect is acheived.
This gives depth to 3D objects
 Brightfield
 use a prism to send the light to both eyes
 light passing through specimen is diffracted and
absorbed to make image
 Staining is often necessary because very low
contrast
microscopy.ppt
Phase Contrast
 A phase ring in condenser allows a cylinder of light through in
phase. Light that is unaltered hits the phase ring in the lens and
is excluded. Light that is slightly altered by passing through
different refractive index is allowed through.
 Light passing through cellular structures such as chromosomes
or mitochondria is retarded because they have a higher
refractive index than the surrounding medium. Elements of
lower refractive index advance the wave. Much of the
backround light is removed and light that constructively or
destructively interfered is let through with enhanced contrast
 Visualizes differences in refractive index of different parts of a
specimen relative to the unaltered light
Hemotoxylin Stain of
Chromosomes (18.5)
Phase Contrast
Differential Interference
Contrast or DIC or Nomarski
 A prism is used to split light into two slightly diverging
beams that then pass through the specimen.
 On recombining the two beams, if they pass through
difference in refractive index then one retarded or
advanced relative to the other and so they can
interfere.
 By changing the prism you can change the beam
separation which can alter the contrast.
 Also measures refractive index changes, but for
narrowly separated regions of light paths-ie it
measures the gradient of RI across the specimen
 Gives a shadowed 3D effect
 Optically sections through a specimen
DIC beam Path
microscopy.ppt
microscopy.ppt
Interference Reflection Microscopy
 Looks at light reflected off the surface only.
 By polarizing the light and then analyzing the
resultant, can see differences in height of
reflecting surface.
 If something is closely opposed to the glass
surface, then it does not pass through a new
medium and when reflected back it is
eliminated.
 Altered light is left in and looks light while
closely apposed is dark.
IRM Light Path
IRM Images
Total Internal Reflection Microscopy
 Light shined on a reflective surface at an
appropriate angle will generate an
evanescent wave, a wave of energy
propagating perpendicular to the surface
 It only propagates about 100-200nm from the
surface
 Allows one to visualize events taking place
near the membrane (exocytosis,
cytoskeleton)
Evanescent Wave
http://www.olympusmicro.com/primer
microscopy.ppt
Specimens
 Live cells or tissue-
 can you see the structure in a live cell?
 can you image the cell without damaging it with light?
 Fixed-try to retain structure intact
 Glutaraldehyde- reacts with amines and cross links them-
destroys 3D structure of many proteins
 Formaldehyde-reacts with amines and cross links them
 slower reaction, reversible, not as extensive
 Methanol, acetone, ethanol, isopropanol- precipitate
material- not as good for retaining structure
 Rapid freeze (liquid helium)- then fix
Fluorescence Microscopy
Fluorescence
Microscopy
 Fluorescent dye- a molecule that
absorbs light of one wavelength and
then re-emits it at a longer
wavelength
 Can be used alone or in
combination with another molecule
to gain specificity (antibodies)
Epifluorescence Microscope
Dead cells stained with a Fluorescent reagent (fluorescent
phalloidin- a fungal toxin) to visualize actin filaments
Endoplasmic Reticulum Stained with a synthetic dye
that dissolves in ER membranes
Brightness of an image
Discussion Problem
 Actin filaments are 8nm in diameter
 We can see a single filament with
phalloidin stain in fluorescence
microscope
 The resolution limit of the microscope is
200nm
 WHY CAN WE VISUALIZE THE
FILAMENT??
Co-localization of Proteins
 FRET- Fluorescence Resonance Energy
Transfer
 If the emission wevelength of one probe overlaps
with the excitation wavelength of another probe
you can get resonance energy transfer
 Non-radiative transfer- the energy is transferred
directly from molecule to molecule
 The two molecules need to be within 10 nm
because the energy transfer falls off with the 6th
power of distance
 You excite with the donor wavelength and
measure emission at the recipient wavelength
Monitor interactions between two proteins. Left: CFP-NIPP1, center:
YFP-PP1, right: FRET. Top: Both YFP-PP1g are expressed. NIPP1 binds
and retargets PP1 to nuclear speckles outside of nucleolus. Bottom:
Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget
speckles from nucleolus. After bleed-through correction, minimal FRET
can be observed (right). Images acquired during 2002 FISH Course
CSHL Labs (Universal Imaging Website).
Co-localization of proteins
 FLIM-Fluorescence Lifetime Imaging
 When a probe is excited briefly, the rate of
decay of fluorescence is different for each
probe-so if you have different probes in the
cell you can characterize them based upon
lifetime
 FRET-FLIM- measure the decay of the
donor during FRET
Confocal Microscopy
 Fluorescence microscope
 Uses “confocality” (a pinhole) to eliminate
fluorescence from out of focus planes
 Minimum Z resolution=0.3µm
 Because you can optically section through a
specimen, you can determine the localization
of probes in the Z dimension
 You can also build 3D (4D) models of
structures and cells from the data
Laser scanning confocal
 Uses a laser to get a high energy point
source of light
 The beam is scanned across the
specimen point by point and the
fluorescence measured at each point
 The result is displayed on a computer
screen (quantitative data)
Laser scanning confocal Microscope
http://www.microscopyu.com
Specimen illumination
Results
Spinning Disk confocal
Microscope
 Illuminates the whole field
“simultaneously with a field of points
 Captures images of the whole field at
once with a camera
 Much faster than LSCM
 Can be viewed through eyepieces
Nipkow spinning disk
Two photon confocal microscopy
 A fluor like fluorescein normally absorbs a photon of about
480nm and emits one at about 530nm
 If fluorescein absorbs two photons of 960nm near enough to
each other in time so that the first does not decay before the
second is absorbed, it will fluoresce- 2 photon fluorescence
 Confocal microscope with a laser that emits picosecond pulses
of light instead of a continuous beam is used
 Advantage
 960nm light penetrates farther into biological specimens
 The density of light is very high at focal point, but low elsewhere, so
damage to cell is less
 You don’t need a second pinhole because excitation only happens
at the focal point
Second harmonic Imaging
 Uses same instrument as 2-photon
microscope
 If you shine 960nm light on a non-
fluorescent sample, interaction of the
light with certain structures will cause it
to be converted to 480nm light
 Works mostly with polarizable materials
like filaments
microscopy.ppt
How do we get fluorescent
probes into cells
 Kill the cell and make the membrane permeable
 Live cells
 Diffusion: some can cross membrane
 Microinjection- stick and tiny needle through membrane
 Trauma: rip transient holes in membrane by mechanical
shear (scrape loading) or electrical pulse
(electroporation)
 Lipid vesicles that can fuse with membrane
 Transfect with fluorescent protein vector
Loading Cells (Alberts 4-59)
Types of Probes
 Some change intensity of fluorescence depending on
pH or [Ca++]
 Some bind specific structures
 ER
 actin
 Golgi
 Plasma membrane
 Mitochondria
 Fluorescently labeled purified protein
 Antibodies
Microinjected Fluorescent Tubulin in a live cell
Immunofluorescence localization of
proteins in dead/fixed cells
 You can purify almost any protein from the
cell (Biochemistry)
 Make an antibody to it by injecting it into a
rabbit or mouse (primary antibody)
 Use the antibody to bind to the protein in the
fixed cell
 Fixed cells can be made permeable so
antibodies can get into interior
 Use a fluorescent “secondary antibody” (anti-
rabbit or mouse) to localize the primary
antibody
Immunofluorescence Visualization of Cell Structures
Anti-tubulin Immunofluorescent localization of microtubules
 Protein from fluorescent jellyfish
 The protein is fluorescent
 Now cloned, sequenced and X-ray structure known
 If you express it in a cell, the cell is now fluorescent!
 Use a liver promoter to drive gene expression, and you get a fluorescent liver! All
cells in the liver make GFP which fills the cytoplasm with fluorescence.
 Fuse the DNA sequence of a protein to the DNA sequence of GFP and the cell
will express it and make a fusion protein which has two domains. Wherever that
protein is in the cell, you will see fluorescence!
 Allows you to do live cell dynamic localization of specific proteins
GFP protein
Green Fluorescent Protein (GFP)- An
Ongoing Revolution in Cell Biology
GFP gene DNA
GFP Protein on Liver
DNA
Liver protein Protein
GFP gene
Liver specific promoter
Liver protein gene
microscopy.ppt
Amoeba cells expressing GFP-Coronin fusion protein (green)
phagocytosing (engulfing and eating) yeast (red)
Indirect visualization of actin filaments-
GFP fusion with an actin binding protein
Problem 2
 I purify a nuclear membrane protein which I
find to be 165kD in size. I then make an
antibody to the protein. When I immunostain
the cell, I get fluorescence in the nuclear
membrane and in the Golgi. When I run a
Western blot, I get a 165kD band and a 60kD
band. Give two explanations to explain the
results and then describe what you would do
to clarify the results.
How to get around the problem of
resolution?
 Invent the Electron Microscope
 Uses electrons instead of light to form an image
 Wavelength of electron decreases as velocity increases so accelerated electrons
have a very short wavelength compared to visible light
 You need to use magnets as lenses to focus the beam
 View electrons striking fluorescent screen
 TEM- Sees electrons that pass through the specimen. Electrons scatter
when they strike the specimen so as density of material increases, more
electrons make it to the detector
 SEM- Looks at the electrons reflected as a beam is passed over the
specimen
 Resolution
 l = 0.004 nm
 If lenses were as good as optical ones, resolution would be 0.002 nm (100,000x better
than light)
 but NA of magnetic lenses is much worse so for biological specimens
 resolution= 2 nm (100x better than light microscope)
microscopy.ppt
microscopy.ppt
microscopy.ppt
Sample Preparation for EM
 Must be done in vacuum for electron gun to work
 Can’t have water in vacuum!
 Dry tissue does not have enough density to scatter electrons so you
have to replace it with something dense.
 Procedure
 Fix Tissue (glutaraldehyde or osmium)
 Dehydrate and embed with plastic
 Stain with Osmium, lead etc. or make metal replica
 For TEM- Section (0.02-0.1µm thick)- so you only look at very thin
section
 For SEM- No sectioning- you only see the outer surface
 What you see is the scattering of electrons by the metal. There is no
biological material left!
microscopy.ppt
microscopy.ppt
microscopy.ppt
Immuno-electron microscopy
 You can’t see antibodies in the EM
 You can attach dense particles to
antibodies to make them visible
 Allows you to visualize the localization
of specific proteins in the EM
 Very hard to do!
microscopy.ppt

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microscopy.ppt

  • 1. Microscopes and Microscopy MCB 380 Good information sources: Alberts-Molecular Biology of the Cell http://micro.magnet.fsu.edu/primer/ http://www.microscopyu.com/
  • 2. Approaches to Problems in Cell Biology  Biochemistry-You can define a enzyme reaction and then try to figure what does it, when, where and under what control  Genetics- You can make a mutation and then try to figure out what you mutated  Cell Biology- You can visualize a process and try to understand it- for instance cell division was one of the earliest  Today- there are no distinctions. You cannot be just one thing, or be knowledgable about one thing. You need to take integrated appoaches to problems using the appropriate tools when needed. If you limit your approach, you limit your science
  • 3. Properties of Light  Reflection  Diffraction-scattering of light around edges of objects  Limits the resolution  Refraction- bending of light when changing medium (index of refraction)  principle that lenses use to focus light  Used in contrasting techniques  Interference  light waves can subtract and add  Polarization- allowing only light of a particular vibrational plane
  • 6. Limitations  light waves diffract at edges-smearing causes limits  resolution = minimum separation of two objects so that they can both be seen
  • 11. Limit of Resolution  The cone of light collected by the lens determines the resolution (nsinq) n=refractive index  Max NA is 1.4 (refractive index of oil)  Lenses range from 0.4-1.4 NA  Maximum magnification is about 1000x Resolution = 0.61l/nsinq = 0.61l/NA
  • 15. Resolution of Microscopes  Visible light is 400-700nm  Dry lens(0.5NA), green(530nm light)=0.65µm=650nm  for oil lens (1.4NA) UV light (300nm) = 0.13µm  for electron microscope  l=0.005nm but NA 0.01 so =30-50nm
  • 16. Sizes of Objects  Eukaryotic cell- 20µm  Procaryotic cell-1-2µm  nucleus of cell-3-5µm  mitochondria/chloroplast- 1-2µm  ribosome- 20-30nm  protein- 2-100nm
  • 17. Microscope Objectives  complex combinations of lenses to achieve  high magnification  low optical distortion  Low chromatic distortion  flat field
  • 18. Contrast  Cells are essentially water and so are transparent  In addition to resolution and brightness, you need to generate contrast to see things  Two objects may be resolvable by the microscope, but if they don’t differ from the background, you cannot see them  Contrast can be accomplished with staining or optical techniques
  • 19. Microscope types  Brightfield  Stereo  Phase contrast  Differential Interference Contrast  Fluorescence  Confocal  Electron  Transmission  Scanning  Atomic Force
  • 20. Microscopes  Stereo  Different images are sent to the two eyes from different angles so that a stereo effect is acheived. This gives depth to 3D objects  Brightfield  use a prism to send the light to both eyes  light passing through specimen is diffracted and absorbed to make image  Staining is often necessary because very low contrast
  • 22. Phase Contrast  A phase ring in condenser allows a cylinder of light through in phase. Light that is unaltered hits the phase ring in the lens and is excluded. Light that is slightly altered by passing through different refractive index is allowed through.  Light passing through cellular structures such as chromosomes or mitochondria is retarded because they have a higher refractive index than the surrounding medium. Elements of lower refractive index advance the wave. Much of the backround light is removed and light that constructively or destructively interfered is let through with enhanced contrast  Visualizes differences in refractive index of different parts of a specimen relative to the unaltered light
  • 25. Differential Interference Contrast or DIC or Nomarski  A prism is used to split light into two slightly diverging beams that then pass through the specimen.  On recombining the two beams, if they pass through difference in refractive index then one retarded or advanced relative to the other and so they can interfere.  By changing the prism you can change the beam separation which can alter the contrast.  Also measures refractive index changes, but for narrowly separated regions of light paths-ie it measures the gradient of RI across the specimen  Gives a shadowed 3D effect  Optically sections through a specimen
  • 29. Interference Reflection Microscopy  Looks at light reflected off the surface only.  By polarizing the light and then analyzing the resultant, can see differences in height of reflecting surface.  If something is closely opposed to the glass surface, then it does not pass through a new medium and when reflected back it is eliminated.  Altered light is left in and looks light while closely apposed is dark.
  • 32. Total Internal Reflection Microscopy  Light shined on a reflective surface at an appropriate angle will generate an evanescent wave, a wave of energy propagating perpendicular to the surface  It only propagates about 100-200nm from the surface  Allows one to visualize events taking place near the membrane (exocytosis, cytoskeleton)
  • 35. Specimens  Live cells or tissue-  can you see the structure in a live cell?  can you image the cell without damaging it with light?  Fixed-try to retain structure intact  Glutaraldehyde- reacts with amines and cross links them- destroys 3D structure of many proteins  Formaldehyde-reacts with amines and cross links them  slower reaction, reversible, not as extensive  Methanol, acetone, ethanol, isopropanol- precipitate material- not as good for retaining structure  Rapid freeze (liquid helium)- then fix
  • 37. Fluorescence Microscopy  Fluorescent dye- a molecule that absorbs light of one wavelength and then re-emits it at a longer wavelength  Can be used alone or in combination with another molecule to gain specificity (antibodies)
  • 39. Dead cells stained with a Fluorescent reagent (fluorescent phalloidin- a fungal toxin) to visualize actin filaments
  • 40. Endoplasmic Reticulum Stained with a synthetic dye that dissolves in ER membranes
  • 42. Discussion Problem  Actin filaments are 8nm in diameter  We can see a single filament with phalloidin stain in fluorescence microscope  The resolution limit of the microscope is 200nm  WHY CAN WE VISUALIZE THE FILAMENT??
  • 43. Co-localization of Proteins  FRET- Fluorescence Resonance Energy Transfer  If the emission wevelength of one probe overlaps with the excitation wavelength of another probe you can get resonance energy transfer  Non-radiative transfer- the energy is transferred directly from molecule to molecule  The two molecules need to be within 10 nm because the energy transfer falls off with the 6th power of distance  You excite with the donor wavelength and measure emission at the recipient wavelength
  • 44. Monitor interactions between two proteins. Left: CFP-NIPP1, center: YFP-PP1, right: FRET. Top: Both YFP-PP1g are expressed. NIPP1 binds and retargets PP1 to nuclear speckles outside of nucleolus. Bottom: Mutant form of CFP-NIPP1. It does not bind PP1, so cannot retarget speckles from nucleolus. After bleed-through correction, minimal FRET can be observed (right). Images acquired during 2002 FISH Course CSHL Labs (Universal Imaging Website).
  • 45. Co-localization of proteins  FLIM-Fluorescence Lifetime Imaging  When a probe is excited briefly, the rate of decay of fluorescence is different for each probe-so if you have different probes in the cell you can characterize them based upon lifetime  FRET-FLIM- measure the decay of the donor during FRET
  • 46. Confocal Microscopy  Fluorescence microscope  Uses “confocality” (a pinhole) to eliminate fluorescence from out of focus planes  Minimum Z resolution=0.3µm  Because you can optically section through a specimen, you can determine the localization of probes in the Z dimension  You can also build 3D (4D) models of structures and cells from the data
  • 47. Laser scanning confocal  Uses a laser to get a high energy point source of light  The beam is scanned across the specimen point by point and the fluorescence measured at each point  The result is displayed on a computer screen (quantitative data)
  • 48. Laser scanning confocal Microscope http://www.microscopyu.com
  • 51. Spinning Disk confocal Microscope  Illuminates the whole field “simultaneously with a field of points  Captures images of the whole field at once with a camera  Much faster than LSCM  Can be viewed through eyepieces
  • 53. Two photon confocal microscopy  A fluor like fluorescein normally absorbs a photon of about 480nm and emits one at about 530nm  If fluorescein absorbs two photons of 960nm near enough to each other in time so that the first does not decay before the second is absorbed, it will fluoresce- 2 photon fluorescence  Confocal microscope with a laser that emits picosecond pulses of light instead of a continuous beam is used  Advantage  960nm light penetrates farther into biological specimens  The density of light is very high at focal point, but low elsewhere, so damage to cell is less  You don’t need a second pinhole because excitation only happens at the focal point
  • 54. Second harmonic Imaging  Uses same instrument as 2-photon microscope  If you shine 960nm light on a non- fluorescent sample, interaction of the light with certain structures will cause it to be converted to 480nm light  Works mostly with polarizable materials like filaments
  • 56. How do we get fluorescent probes into cells  Kill the cell and make the membrane permeable  Live cells  Diffusion: some can cross membrane  Microinjection- stick and tiny needle through membrane  Trauma: rip transient holes in membrane by mechanical shear (scrape loading) or electrical pulse (electroporation)  Lipid vesicles that can fuse with membrane  Transfect with fluorescent protein vector
  • 58. Types of Probes  Some change intensity of fluorescence depending on pH or [Ca++]  Some bind specific structures  ER  actin  Golgi  Plasma membrane  Mitochondria  Fluorescently labeled purified protein  Antibodies
  • 60. Immunofluorescence localization of proteins in dead/fixed cells  You can purify almost any protein from the cell (Biochemistry)  Make an antibody to it by injecting it into a rabbit or mouse (primary antibody)  Use the antibody to bind to the protein in the fixed cell  Fixed cells can be made permeable so antibodies can get into interior  Use a fluorescent “secondary antibody” (anti- rabbit or mouse) to localize the primary antibody
  • 63.  Protein from fluorescent jellyfish  The protein is fluorescent  Now cloned, sequenced and X-ray structure known  If you express it in a cell, the cell is now fluorescent!  Use a liver promoter to drive gene expression, and you get a fluorescent liver! All cells in the liver make GFP which fills the cytoplasm with fluorescence.  Fuse the DNA sequence of a protein to the DNA sequence of GFP and the cell will express it and make a fusion protein which has two domains. Wherever that protein is in the cell, you will see fluorescence!  Allows you to do live cell dynamic localization of specific proteins GFP protein Green Fluorescent Protein (GFP)- An Ongoing Revolution in Cell Biology GFP gene DNA GFP Protein on Liver DNA Liver protein Protein GFP gene Liver specific promoter Liver protein gene
  • 65. Amoeba cells expressing GFP-Coronin fusion protein (green) phagocytosing (engulfing and eating) yeast (red)
  • 66. Indirect visualization of actin filaments- GFP fusion with an actin binding protein
  • 67. Problem 2  I purify a nuclear membrane protein which I find to be 165kD in size. I then make an antibody to the protein. When I immunostain the cell, I get fluorescence in the nuclear membrane and in the Golgi. When I run a Western blot, I get a 165kD band and a 60kD band. Give two explanations to explain the results and then describe what you would do to clarify the results.
  • 68. How to get around the problem of resolution?  Invent the Electron Microscope  Uses electrons instead of light to form an image  Wavelength of electron decreases as velocity increases so accelerated electrons have a very short wavelength compared to visible light  You need to use magnets as lenses to focus the beam  View electrons striking fluorescent screen  TEM- Sees electrons that pass through the specimen. Electrons scatter when they strike the specimen so as density of material increases, more electrons make it to the detector  SEM- Looks at the electrons reflected as a beam is passed over the specimen  Resolution  l = 0.004 nm  If lenses were as good as optical ones, resolution would be 0.002 nm (100,000x better than light)  but NA of magnetic lenses is much worse so for biological specimens  resolution= 2 nm (100x better than light microscope)
  • 72. Sample Preparation for EM  Must be done in vacuum for electron gun to work  Can’t have water in vacuum!  Dry tissue does not have enough density to scatter electrons so you have to replace it with something dense.  Procedure  Fix Tissue (glutaraldehyde or osmium)  Dehydrate and embed with plastic  Stain with Osmium, lead etc. or make metal replica  For TEM- Section (0.02-0.1µm thick)- so you only look at very thin section  For SEM- No sectioning- you only see the outer surface  What you see is the scattering of electrons by the metal. There is no biological material left!
  • 76. Immuno-electron microscopy  You can’t see antibodies in the EM  You can attach dense particles to antibodies to make them visible  Allows you to visualize the localization of specific proteins in the EM  Very hard to do!